Endotoxins (lipopolysaccharides) are thought to play a major role in the pathogenesis of invasive gram-negative bacterial diseases; the infected host's inflammatory reaction to these molecules may produce considerable morbidity and even death. Little is known about host mechanisms for detoxifying lipopolysaccharides (LPS). During the past four years we identified a novel human enzyme that cleaves certain fatty acids from LPS and showed that this enzymatic deacylation greatly modifies the bioactivities of LPS. We now propose to evaluate the biological significance of this enzyme in man. Lipid A, the bioactive moiety of LPS, is a glucosamine disaccharide that has covalently linked fatty acids, phosphates, and a polysaccharide chain. Hydroxylated fatty acids (e.g., 3- hydroxymyristate) are linked directly to glucosamine, whereas normal fatty acyl chains (myristoyl and lauroyl) are substituted to the hydroxyl groups of some of the 3-hydroxymyristoyl residues to form acyloxyacyl linkages. We found an enzymatic activity in human neutrophils that removes (only) the normal fatty acids from LPS--breaking the acyloxyacyl linkages. This acyloxyacyl hydrolysis was then shown to reduce the bioactive potency of LPS. Interestingly, the impact of enzymatic deacylation was greater on the tissue toxicity of LPS than on the immunostimulatory activity, suggesting that the enzyme may act in vivo to reduce LPS toxicity while retaining potentially beneficial stimuli to the immune system. The proposed research will (1) complete the purification and characterization of human neutrophil acyloxyacyl hydrolase (AOAH), (2) clone the gene for AOAH, (3) determine the cellular location(s) of AOAH and evaluate its regulation in cultured cells and in vivo, and (4) use deacylated LPS (which inhibit the ability of LPS to stimulate human endothelial cells) to study the stimualtion of cells by LPS. Acyloxyacyl hydrolase is the only enzyme that is known to interact with LPS in animal cells. We hope that understanding its biological role(s) will suggest new strategies for controlling inflammatory responses to LPS and consequently improve the outcome of gram-negative bacterial diseases in man.